Monocyte-mediated regulation of genes by the amyloid and ... · Beatriz Morte, Tamara Martínez,...
Transcript of Monocyte-mediated regulation of genes by the amyloid and ... · Beatriz Morte, Tamara Martínez,...
Monocyte-mediated regulation of genes by the amyloid and prion peptides in SH-SY5Y neuroblastoma cells Beatriz Morte, Tamara Martínez, Alberto Zambrano and Angel Pascual Instituto de Investigaciones Biomédicas. Consejo Superior de Investigaciones Científicas. Madrid. Spain. Running Title: Upregulation of genes by amyloid and prion peptides
Address correspondence: Dr. A. Pascual Instituto de Investigaciones Biomédicas. (C.S.I.C.). Arturo Duperier, 4 28029 Madrid. Spain. Tel. 34-91-585 4460 Fax. 34-91-585 4401 e-Mail: [email protected]
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ABSTRACT
Alzheimer’s disease as well as prion-related encephalopathies are
neurodegenerative disorders of the central nervous system, which cause
mental deterioration and progressive dementia. Both pathologies appear to be
primarily associated with the pathological accumulation and deposit of -
amyloid or prion peptides in the brain, and it has been even suggested that
neurotoxicity induced by these peptides would be associated to essentially
similar pathogenic mechanisms, in particular to those that follow the activation
of microglial cells. To probe whether the neurotoxic effects induced by the -
amyloid and prion peptides are actually mediated by similar glial-associated
mechanisms, we have examined the differential expression of genes in SH-
SY5Y neuroblastoma cells incubated with conditioned media from -amyloid or
prion-stimulated THP-1 monocytic cells. According to microarray analysis, not
many coincidences are observed and only four genes (Hint3, Psph, Daam1 and
c-Jun) appear to be commonly upregulated by both peptides. Furthermore, c-
Jun appears to be involved in the cell death mediated by both peptides.
INTRODUCTION
Alzheimer and prion pathologies are neurodegenerative diseases
characterized by the progressive loss of neuronal synaptic function, an effect
that in both cases appears to be secondary to the toxicity induced by key
proteins, the beta-amyloid (A) in Alzheimer’s disease and PrP in prion
pathologies, which accumulate and aggregate in the brain. At first sight, both
types of pathologies exhibit different specific characteristics in relation with the
mode of transmission, the etiology, or the frequency of occurrence, and they
can be considered as essentially distinct pathologies (Checler & Vincent, 2002).
However, despite these evident differences, these pathologies also show a
number of similarities. In both cases neurodegeneration appears to be
mediated by proteins that, as mentioned above, are aggregated and
accumulated in the brain (Price et al., 1993). Those proteins share a complex
multi-domain structure and contain toxic sequences that mediate neurotoxicity
and give rise to similar damaging apoptotic phenotypes (Checler & Vincent,
2002). Moreover, they share a number of properties such as the presence of
metal-binding sites that mediate the binding of metals ions, in particular copper
or zinc (Barnham et al., 2006), or the existence of several repeats of a GxxxG
motif in the transmembrane region, which contain a methionine residue that
appears to be essential to modulate the neurotoxicity of A in Alzheimer or the
disease susceptibility in the prion associated pathologies (Barnham et al.,
2006). Evidence reported in those articles supported the hypothesis that
neurotoxicity induced by both proteins, the amyloid and prion, would be
mediated by similar, if not identical, molecular pathways and opened a very
attractive possibility as is the use of common therapeutic strategies to treat and
prevent Alzheimer’s and prion diseases.
Unfortunately, this hypothesis appears to be too simplistic, and although it
is evident that both the -amyloid and the PrP proteins are neurotoxic and
induce similar processes of apoptosis and neurodegeneration, the factors and
pathways that mediate these effects could be essentially different (Forloni et al.,
1996; Hope et al., 1996; Brown et al., 1997). In this sense, we have also
reported that both the amyloid-beta 25-35 (A25-35) and the PrP106-126
fragments induce a distinct gene expression profile in the human SH-SY5Y
neuroblastoma cell line. First, the number of genes significantly altered by
these treatments was markedly different, 198 in the case of the prion (Martinez
& Pascual, 2007b), and 67 in the case of the amyloid fragment (Martinez &
Pascual, 2007a), and second, the relationship between the overexpressed and
repressed genes was opposite in both cases (16/182 and 54/13, respectively).
Despite this, it has been also well established that in addition to those
effects directly induced on neurons, neurotoxicity of both peptides can be
largely mediated by microglia activation, and the consequent release of
cytotoxic molecules such as proinflammatory cytokines or reactive oxygen
intermediates. In fact, an increasing body of evidence indicates that neuronal
death induced by the -amyloid and the prion peptides in the brain is mainly
associated to processes that are promoted by activation of the surrounding
microglial cells (Dheen et al., 2007). To analyze possible coincidences between
the neurotoxic effects induced by both peptides through activation of microglia,
we have now analyzed the gene expression profile in SH-SY5Y neuroblastoma
cells exposed to conditioned media obtained from A25-35 or PrP106-126
stimulated THP-1 human monocytic cells, which were used as a surrogate
model of human microglia.
MATERIALS AND METHODS
Cell Culture and treatments
Human monocytic THP-1 cells and SH-SY5Y human neuroblastoma cells
were cultured as previously described (Combs et al., 1999; Villa et al., 2002) in
RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco.Life
technologies Ltd. Paislay. Scotland. UK), and maintained at 37ºC in a 5% CO2
atmosphere.
Synthetic A25-35 obtained from AnaSpec and synthetic PrP106-126
obtained from Neosystem, were prepared as follows: Briefly, A25-35 was
dissolved in deionized-distilled water at a concentration of 2,5 mM and stored at
−80 °C. Previous to the experiments, the stock solution was diluted to the
desired concentrations and then added to the culture medium. The prion
peptide 106-126 was dissolved in PBS at a concentration of 2 mM and
maintained for 48 hours at room temperature to allow polymerization, before
being added to the cells.
For experiments, THP-1 monocytes were incubated for 48 hours with or
without 10 M A25-35 or 10M PrP106-126, and the conditioned media
collected and centrifuged to eliminate cell residues. The cell-free supernatants
were then used to replace the culture media of SH-SY5Y cells previously plated
in 6-well plates and grown for 24 hours in RPMI/10%FBS media. Six hours after
addition of conditioned media a set of cells was harvested for posterior RNA
extraction and determination of gene expression. The remaining cells were
incubated in the presence or in the absence of conditioned media for 24 hours
and then photographed and fixed for histological analysis or collected and
saved for subsequent protein or RNA extraction.
Fluorescence microscopy
SH-SY5Y cells were incubated for different time periods with the
supernatants of amyloid- or prion-treated THP-1 cells or RPMI fresh medium,
and then fixed with 4% paraformaldehyde at pH=7,4 and room temperature for
1 hour, permeabilized with 0,1% Triton-X-100, and then subjected to TUNEL
assay to evaluate the cellular death by using the in situ cell death detection kit
Fluorescein (Roche). To further confirm the findings of TUNEL assay, cells
were washed with PBS, stained with DAPI, and analyzed by fluorescence
microscopy, using a Zeiss Axiophot inverted fluorescence microscope (515-565
nm for TUNEL, and 450-480 nm for DAPI). Images of cells were then digitized
using an Olympus DP70 color camera.
MTT cell viability assay
Cell viability was evaluated in 96-well culture plates using a colorimetric
assay based on the reduction of tetrazolium dye (MTT) to a blue formazan
product. After incubation for 4 h with MTT (0.5 mg/ml) at 37°C, isopropanol/HCl
was added to each 96-well and the absorbance of solubilized MTT formazan
products was measured spectrophotometrically at 570 nm.
RNA interference
Expression of c-Jun in the SH-SY5Y cells was inhibited by using the
Santa Cruz Biotechnology c-Jun siRNA (h2) (catalog number sc-44201). Cells
were grown to 60% confluence and the c-Jun siRNA or a control siRNA (catalog
number sc-37007) were transfected (using the transfection reagent sc-29528)
according to the manufacturer’s instructions. At 24 h after transfection, the
medium was replaced and the cells were further incubated for 48 h. Next, media
were removed and the cells were exposed to the THP-1 conditioned
supernatants for 24 h.
RNA preparation and Microarray Analysis
For RNA isolation cell lysates were homogenized and the RNA purified by
using the QIAshredder and RNeasy Mini kits of Qiagen, according to the
manufacturer's recommendations. The final amount of isolated RNA was
determined in each sample by spectrophotometry and its quality assessed by
electrophoresis on agarose gels.
Preparation of cDNA, cRNA, hybridization and scanning of microarrays
were performed following manufacturer’s protocols. cDNA, and biotinylated
cRNAs were synthesized from 5g RNA samples with the GeneChip
expression 3’ amplification reagents (one-cycle cDNA synthesis, and IVT
labelling) kits of Affymetrix, and biotinylated probes were hybridized to an
Affymetrix gene chip human genome U133A Plus 2.0 array, a microarray that
contains more than 54,000 probe sets and allows an accurate analysis of the
quantitative expression of over 47,000 transcripts, including 38,500 well
characterized human genes. Analysis for differential expression was performed
using the R platform for statistical analysis (R Foundation for Statistical
Computing. Vienna) and several packages from the Bioconductor project
(Gentleman et al., 2004; Carey et al., 2005). The raw data were imported into R
and pre-processed using the affy package and the robust multichip average
method (Irizarry et al., 2003). Genes were selected based on fold change. For
this task an absolute fold change of 1.6 was used.
RT-PCR amplification of mRNA
RT-PCR was used to validate the differential expression of several A- or
PrP-responsive genes detected in the microarray analysis. Total RNA was
extracted from cell cultures as mentioned above and cDNA was prepared from
250 ng of RNA using the high-capacity cDNA reverse transcription kit (Applied
Biosystems, Foster City, CA). For quantitative PCR, a cDNA aliquot
corresponding to 5 ng of the starting RNA was used, with Taqman Assay-on-
Demand primers and the Taqman universal PCR master mix, No Amp Erase
UNG (Applied Biosystems) on a 7900HT fast real-time PCR system (Applied
Biosystems). The PCR program consisted in a hot start of 95 C for 10 min,
followed by 40 cycles of 15 sec at 95 C and 1 min at 60 C. PCRs were
performed in triplicates, using the 18S gene as internal standard and the 2-
cycle threshold method for analysis (Livak & Schmittgen, 2001).
Statistical analysis.
When appropriate, the significance of differences was calculated with the
Student's t test, and it was indicated in the corresponding figure by the following
symbol: * p < 0,05 and ** p< 0.01.
RESULTS
Effects of the THP-1 conditioned medium on neuronal morphology and
viability.
It is widely accepted that both the -amyloid and prion peptides induce
microglial activation and the subsequent secretion of cytokines and neurotoxic
reactive oxygen species (Forloni et al., 1993; Klegeris et al., 1997), which in
turn may increase neuronal apoptosis (Dheen et al., 2007).
To further analyze and compare how the -amyloid- and prion-stimulated
microglia may affect neurons, we have exposed SH-SY5Y neuroblastoma cells
to conditioned media obtained from THP-1 cells, a monocytic cell line that
exhibits responses to stimuli similar to those of microglia (Combs et al., 1999).
THP-1 cells were incubated with or without 1,2 5 or 10M A25-35 or PrP106-
126 for a 48 h time period and the recovered conditioned media was added to
SH-SY5Y neuroblastoma cells that were then incubated for an additional 24
hours time period. The morphology and viability of cells were estimated by
optical microscopy, TUNEL and DAPI staining (figure 1), and by MTT assay
and Western blot analysis of caspase-3 (figure 2).
As illustrated in figure 1, morphology and viability of cells were not
affected by conditioned medium from THP-1 cells treated with scrambled
sequences, with 5M A25-35 or PrP106-126 (panel A), or with lower doses of
those peptides. However, the cell morphology as well as the viability appeared
to be severely affected after 24 hours of exposition to conditioned media from
10M A25-35 or PrP106-126 treated THP-1 cells (panel B). As shown in this
panel, a large amount of neuroblastoma cells become apoptotic after 24 hours
incubation with conditioned media from 10 M A25-35 or PrP106-126 treated
cells. The number of cells was clearly reduced by both treatments and the
residual cells became more rounded in appearance. Figure 2 includes the
results obtained in a representative MTT assay (panel A), which confirms the
reduced viability of cells, and the levels of activated caspase-3 detected by
Western blot (panel B), which corroborated that apoptosis was significantly
increased in SH-SY5Y cells incubated with those supernatants obtained from
amyloid- or prion-stimulated THP-1 cells. All together, these results evidence
that cell death was drastically increased in SH-SY5Y cells exposed for 24 hours
to conditioned media recovered from amyloid- or prion-stimulated THP-1 cells.
Gene expression profile in SH-SY5Y cells incubated in THP-1 cells-
conditioned culture media.
To analyze how the amyloid- and prion-activated microglia may affect the
gene expression profiling in neurons, SH-SY5Y cells were exposed to
conditioned media for a 6 hours time period and then harvested and saved for
posterior RNA extraction and microarray analysis. Gene expression was
analyzed by using an Affymetrix U133A plus2 array and the data analyzed as
described under Materials and Methods.
As expected, both the amyloid- and prion-conditioned media induced
changes on the gene expression profile of treated SH-SY5Y cells. A
significance analysis of microarrays highlighted a relatively high number of
transcripts differentially expressed in cells exposed to conditioned media from
the THP-1 monocytic cells stimulated by the amyloid or prion fragments.
However, the intensity of the response was rather low and not many genes
change their expression by more than 1.6-fold. Of interest, the gene expression
profiles appear to be different in both cases. First, the number of genes that
change their expression appears to be much higher in amyloid-treated cells
than in those cells exposed to the prion-conditioned medium. Second, whereas
the genes affected by the prion-induced media appear to be mainly
upregulated, the genes differentially expressed in cells exposed to the amyloid-
conditioned media were largely down-regulated. These results are illustrated in
the Venn diagram shown in figure 3, which outlines the total number of
sequences up- and down-regulated, as well as the number of coincidences,
detected in cells exposed to both treatments. As illustrated in this figure, a total
of 168 sequences, 19 upregulated and 149 down-regulated, in cells exposed to
the amyloid peptide-conditioned media, and a total of 17 sequences, all of them
upregulated, in the cells treated with the prion-conditioned media, were found to
be differentially expressed (above 1,6 fold with respect to control). After
subtracting non annotated sequences and repeated probes the number of
amyloid- and prion-induced genes were reduced to 141 (129 down- and 12
upregulated) and 11 (all of them upregulated), respectively. Furthermore, four
of these sequences appeared to be commonly upregulated by both treatments.
The lists of the differentially expressed transcripts has been included in
tables 1 (amyloid) and 2 (prion), which, as mentioned before, only include
transcripts showing a 1,6-fold variation above or below the level found in the
control.
Validation of the microarray data
To confirm the results obtained in the microarray analysis, the
expression of a subset of genes selected from those identified in the microarray
was analyzed by real-time PCR performed with cDNAs prepared from the
RNAs of SH-SY5Y cells treated, or untreated, with conditioned media
recovered from prion- or amyloid-stimulated THP-1 cells.
Transcripts chosen for confirmation included a total of 9 genes selected
among those commonly deregulated by both the prion- and amyloid-
conditioned media and/or because of their involvement in development and
plasticity of the nervous system. As expected, the results from real-time PCR
analysis were comparable to those provided by microarray analysis and,
although in some of the samples the intensity of the response was different to
that expected, all the transcripts analyzed showed the predicted differential
expression patterns. Figure 4 shows the results obtained for five of these
genes, two of them (JUN and DAAM1) commonly regulated by both treatments,
and other three genes (STMN4, CREB5, and ETNK1) selected because of their
contribution to the Jun-dependent signaling pathways. In agreement with the
microarray data, four of these genes appear to be upregulated by both
treatments, whereas ETNK1 that is down regulated in the amyloid treated cells
remains unchanged in cells treated with the prion-stimulated medium. In
addition, the differences obtained with the other analyzed genes (HINT3,
CREB1, BCLAF1 and MAP3K) were not significant, but also showed the same
tendency observed in the microarrays (not shown).
c-Jun silencing decreases the monocytic-mediated cell death
To further prove the role of c-Jun in the apoptotic signals induced
by the amyloid and prion peptides, we also analyzed the levels of activated
caspase-3 and the cell viability in SH-SY5Y cells transfected with a specific c-
Jun small interference RNA (c-Jun siRNA), which reduced c-Jun levels by
about 50-60% (Figure 5). As shown in the panel A of this figure, the levels of
cleaved caspase-3 were, as expected, increased in control cells transfected
with a non-specific siRNA and exposed to supernatants recovered from the
prion- or amyloid-stimulated THP-1cells. However, a significant decrease in
caspase-3 cleavage was observed in SH-SY5Y cells transfected with the
specific c-Jun siRNA, thus suggesting a role for this protein in the apoptotic
pathways induced by both peptides. In addition, cell viability (MTT assay) was
not significantly affected by c-Jun depletion in the absence of the peptide
fragments, but the reduction of cell viability induced by the A- or PrP-
stimulated conditioned media was stronger in the c-Jun expressing cells than in
the in the siRNA-transfected cells (panel B), further supporting a role for c-Jun
in those processes.
DISCUSSION
Neurodegenerative diseases, in particular Alzheimer´s disease and prion
associated pathologies have been shown to share a number of characteristics
and it has been even suggested that neurotoxicity induced by both, the amyloid
and prion proteins, would be mediated by similar, if not identical, molecular
pathways and mechanisms. In this sense, several factors have been already
pointed as common targets of A and prions (Ferreiro et al., 2006; Ferreiro et
al., 2007; Lopes et al., 2007), and common therapies based on these
descriptions have been suggested (Checler & Vincent, 2002; Barnham et al.,
2006). In contrast, a number of discordances have been also described. As
shown in a number of in vitro studies, A and PrP peptides appear to induce
neuronal apoptosis through different mechanisms (Forloni et al., 1996; Hope et
al., 1996) (Brown et al., 1997). Also in this line, we have described different
gene expression profiles in neuroblastoma cells directly exposed to A25-35
and PrP106-126 synthetic fragments (Martinez & Pascual, 2007b; a). However,
in these reports we just described the effects directly induced by the prion and
amyloid proteins on neurons, whereas neurotoxicity of both peptides has been
proved to be further enhanced by the presence of glial (mostly microglial) cells
(Dheen et al., 2007). In fact, the number of glial cells has been described to
correlate with the amyloid or prion deposition (Van Everbroeck et al., 2004),
and it has been suggested that activation of microglia by A or PrP and the
subsequent release of cytokines, reactive oxygen species and other neurotoxic
factors would be the main cause of the enhanced apoptosis and neuronal death
observed both in Alzheimer and prion diseases. To analyze possible
coincidences in the microglia-mediated neuronal response to those peptides we
have exposed neuroblastoma SH-SY5Y cells to conditioned media obtained
from A- or PrP-stimulated THP-1 monocytic cells, a established cell line widely
used as a model for human microglia because of their functional similarities
with primary microglia and their ability to activate similar signalling pathways
(McDonald et al., 1997; Combs et al., 1999). As expected, exposure of
neuroblastoma cells to conditioned media from A or PrP-treated THP-1 cells
caused a significant increase of cell death that was clearly detectable by optical
microscopy, and further proved by TUNEL staining and caspase-3 activation. In
addition, we have also identified a number of genes whose expression appears
to be deregulated in cells exposed to conditioned media. In both cases, it is
noteworthy that amyloid-associated effects were always more evident that
those induced by the prion-stimulated medium. First, the A25-35 conditioned
media appeared to be more toxic than that conditioned by PrP106-126 and
apparently caused a higher lethality, as observed by optical microscopy.
Second, in contrast to that observed when these peptides act directly on
neurons (Martinez & Pascual, 2007b; a), the number of genes deregulated by
A25-35 was markedly higher than the number of genes affected by the prion-
conditioned media. Indeed, whereas A25-35-induced medium changes the
expression of 141 genes by more than 1,6-fold with respect to the control
group, only 11 genes were altered by the PrP106-126 fragment.
On the other hand, a comparative analysis of these results with those
previously described in neuroblastoma cells directly exposed to the amyloid or
prion fragments, shows a number of additional differences between the direct
and macrophage-mediated effects induced by both peptides. So, whereas the
amyloid fragment appears to activate similar pathways and processes both
acting directly on neurons or indirectly through monocytic activation, the effects
of the PrP106-126 peptide are largely different when exerted directly or
indirectly on neurons.
Despite these marked differences, it is remarkable the common activation
of four genes in cells exposed to both treatments; Hint3, which has not been
previously associated to neurodegenerative pathologies; Psph that is widely
expressed in neuronal ganglia and Daam1 and c-Jun, which as previously
described play a role in the developing and injured nervous system (Kida et al.,
2004; Zhou et al., 2004). In particular, it results of interest the macrophage-
mediated upregulation of c-JUN, a gene that has been associated to axon
regeneration (Raivich et al., 2004; Zhou et al., 2004), to brain inflammation and
to a wide number of processes involved in neuronal cell death and
degeneration (Raivich, 2008). Moreover, a direct relationship between the
JNK/c-Jun cascade and the Alzheimer’s disease (Sun et al., 2009) or prion-
related pathologies (Lee et al., 2005) has been suggested. In the present study
the role of c-Jun in the apoptotic processes induced by the A25-35 and
PrP106-126 peptides has been confirmed by the reduced levels of cleaved
caspase-3, an apoptotic marker, and the partial reversal of the cell viability
reduction found in SH-SY5Y cells depleted of c-Jun by means of a specific
siRNA.
In conclusion, our data show a number of divergences between the genes
and mechanisms that mediate the neurotoxicity of A and PrP peptides but,
remarkably, also show the common activation of a reduced number of genes
including c-Jun, which codes for a transcription factor largely involved in
apoptotic processes. In this sense, PCR analysis also shows the upregulation of
Creb5 a member of the CRE (cAMP response element)-binding protein family,
which as previously reported (Dong et al., 2007) may specifically interact with c-
Jun and function as a CRE-dependent transactivator. These results further
confirm previous descriptions about the role of c-Jun in neurodegenerative
processes and reinforce the JNK/c-Jun pathway as a valuable target for the
development of common therapeutic strategies. However, it is also true that our
experiments have been carried out using two established cell lines as models,
and it could occur that they do not accurately reflect the microglial-mediated
neuronal toxicity that both the amyloid and prion peptides exert in vivo. In any
case, these results might contribute to increase the knowledge about the
mechanisms that mediate the neurotoxicity of both the amyloid and the prion
peptides and should be useful to develop new and specific multitargeted
therapies for these diseases.
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Villa, A., Santiago, J., Garcia-Silva, S., Ruiz-Leon, Y. & Pascual, A. (2002)
Serum is required for release of Alzheimer's amyloid precursor protein in neuroblastoma cells. Neurochem Int, 41, 261-269.
Zhou, F.Q., Walzer, M.A. & Snider, W.D. (2004) Turning on the machine:
genetic control of axon regeneration by c-Jun. Neuron, 43, 1-2.
LEGEND TO THE FIGURES Figure 1. Neurotoxic effects of the conditioned medium from cells treated with
the 25–35 -amyloid and 106–126 PrP fragments. SH-SY5Y cells were cultured
for 24 h in the absence (control) or in the presence of conditioned media from
-amyloid- or prion-treated THP-1 monocytic cells, and the cell death was
assessed by optical microscopy, DAPI staining and TUNEL analysis. Panel A
illustrates results obtained with conditioned media from THP-1 cells treated with
5M A25–35 or PrP106–126 (left), or with 10M of the corresponding
scrambled peptides (right). Panel B shows the effects of conditioned media
from THP-1 cells exposed to 10M A25–35 or PrP106–126.
Figure 2. Viability of SH-SY5Y cells incubated with or without THP-1
conditioned media. Cell death was evaluated by MTT assay or Western blot
detection of activated caspase-3. Panel A illustrates the results obtained in a
representative MTT assay performed in quadruplicate. Data (mean ± S.D) are
expressed relative to the values obtained in control cells **P < 0.01. Panel B
shows the Western blot determination of cleaved caspase-3 and -tubulin
(loading control).
Figure 3. Venn diagram showing transcripts distribution between the amyloid
and prion treated cells. The diagram was generated from lists of transcripts that
are ≥1.6-fold up- (↑) or down- (↓) regulated by both treatments.
Figure 4. Validation of microarray data by quantitative reverse transcriptase
polymerase chain reaction (RT-PCR). The differential expression of some
genes identified in the microarray assay was analyzed by quantitative RT-PCR
carried out with RNA obtained from cells treated for 6 h with or without A25-35-
or PrP106–126-conditioned media. Data are expressed as fold change relative
to control values obtained in untreated cells. *P < 0.05, **P < 0.01.
Figure 5. Silencing of c-Jun protects cells against A and PrP toxicity. SH-SY5Y
cells, transfected with a control (left) or a c-Jun specific siRNA (right), were
incubated for 24 hours with conditioned media from -amyloid or prion-
stimulated THP-1 cells. Panel A illustrates the Western blot determination of c-
Jun, cleaved caspase-3 and -tubulin (loading control). Panel B illustrates the
results obtained in a MTT assay performed in quadruplicate. Data (mean ± S.D)
are expressed relative to the values obtained in cells transfected with the
control siRNA in the absence of peptide fragments. **P < 0.01
Table 1. Deregulated genes in SH-SY5Y cells incubated for 6 hours with media
conditioned by A25-35-treated THP-1 cells. Table includes up and
downregulated genes that change their expression by more than 1.6-fold with
respect to the control group (Nonannotated or repeated genes have been
excluded of this table).
Table 2. Deregulated genes in SH-SY5Y cells incubated for 6 hours with media
conditioned by PrP106-126-treated THP-1 cells. Table includes upregulated
genes that change their expression by more than 1.6-fold with respect to the
control group. Not genes were found decreasing their expression by more than
1,6-fold. List does not included non annotated or repeated genes.
Control
A25-35 Scrambled A25-35
PrP106-126 Scrambled PrP106-126
Control
A25-35
PrP106-126
DAPI TUNEL
A
B
Figure 1
MTT
rela
tive
units
0C Aβ
0,5
1
PrP
Caspase-3
α-tubulin
C Aβ PrP
A
B
**
**
Figure 2
PrP106-126 Aβ25-35
13 4 15
14900
Figure 3
STMN4 CREB5 JUN DAMM1 ETNK10
1
2
3
4
5controlPrP106-126Aβ25 -35
Fold
Cha
nge
**
** **
**
***
ns * ns ns
Figure 4
Cleaved caspase-3
α-tubulin
c-Jun
- Aβ PrP - Aβ PrP
c-Jun siRNAControl siRNA
B
MTT
rela
tive
units
0Aβ
0,5
1
PrP
A
c-Jun siRNA
-- Aβ PrP
** **
Control siRNA
Figure 5
Table 1. Deregulated genes in Aβ25-35-treated cells
Downregulated genes
Gene symbol Gene name Fold change
BCLAF1 BCL2-associated transcription factor 1 -2.35TWF1 twinfilin, actin-binding protein, homolog 1 (Drosophila) -2.31GNA13 guanine nucleotide binding protein (G protein), alpha 13 -2.3SCD stearoyl-CoA desaturase (delta-9-desaturase) -2.27MALAT1 metastasis associated lung adenocarcinoma transcript 1 -2.2SKP2 S-phase kinase-associated protein 2 (p45) -2.19ANKRD10 ankyrin repeat domain 10 -2.17CDK6 cyclin-dependent kinase 6 -2.17CEP170L centrosomal protein 170kDa-like -2.13TAF9B TAF9B RNA polymerase II, TATA box binding protein (TBP)-associated -2.13XIST X (inactive)-specific transcript (non-protein coding) -2.1DCUN1D1 DCN1, defective in cullin neddylation 1, domain containing 1 -2.08PTPN12 protein tyrosine phosphatase, non-receptor type 12 -2.08COPA coatomer protein complex, subunit alpha -2.07SUPT16H suppressor of Ty 16 homolog (S. cerevisiae) -2.07PIK3C2A phosphoinositide-3-kinase, class 2, alpha polypeptide -2.06PPAT phosphoribosyl pyrophosphate amidotransferase -2.06SMC3 structural maintenance of chromosomes 3 -2.06SCD stearoyl-CoA desaturase (delta-9-desaturase) -2.04EXOC5 exocyst complex component 5 -2.02SLC39A6 solute carrier family 39 (zinc transporter), member 6 -2NUCKS1 nuclear casein kinase and cyclin-dependent kinase substrate 1 -1.99GULP1 GULP, engulfment adaptor PTB domain containing 1 -1.97PHTF2 putative homeodomain transcription factor 2 -1.97CBX5 chromobox homolog 5 (HP1 alpha homolog, Drosophila) -1.95CLIC4 chloride intracellular channel 4 -1.95CDC27 cell division cycle 27 homolog (S. cerevisiae) -1.89LOC100190986 hypothetical LOC100190986 -1.89MEX3C mex-3 homolog C (C. elegans) -1.89TMX1 thioredoxin-related transmembrane protein 1 -1.89YTHDF3 YTH domain family, member 3 -1.88EIF4A2 eukaryotic translation initiation factor 4A, isoform 2 -1.87TMEM30A transmembrane protein 30A -1.87BRCC3 BRCA1/BRCA2-containing complex, subunit 3 -1.85EXOC4 exocyst complex component 4 -1.85GTF2I general transcription factor II, i -1.85JAM3 junctional adhesion molecule 3 -1.85PNRC2 proline-rich nuclear receptor coactivator 2 -1.85SCAMP1 secretory carrier membrane protein 1 -1.85ZNF24 zinc finger protein 24 -1.85C5orf22 chromosome 5 open reading frame 22 -1.84CREB1 cAMP responsive element binding protein 1 -1.84LARP4 La ribonucleoprotein domain family, member 4 -1.84USP10 ubiquitin specific peptidase 10 -1.84DHX9 DEAH (Asp-Glu-Ala-His) box polypeptide 9 -1.83MCL1 myeloid cell leukemia sequence 1 (BCL2-related) -1.83NUDT21 nudix (nucleoside diphosphate linked moiety X)-type motif 21 -1.83CD46 CD46 molecule, complement regulatory protein -1.82ACTR2 ARP2 actin-related protein 2 homolog (yeast) -1.8SLC16A1 solute carrier family 16, member 1 (monocarboxylic acid transporter 1) -1.8
Table1
SRPK1 SFRS protein kinase 1 -1.8VPS35 vacuolar protein sorting 35 homolog (S. cerevisiae) -1.8BCAT1 branched chain aminotransferase 1, cytosolic -1.8C10orf18 chromosome 10 open reading frame 18 -1.79FYTTD1 forty-two-three domain containing 1 -1.79WAC WW domain containing adaptor with coiled-coil -1.79CPD carboxypeptidase D -1.78IGF2BP3 insulin-like growth factor 2 mRNA binding protein 3 -1.78MTPAP mitochondrial poly(A) polymerase -1.78G3BP2 GTPase activating protein (SH3 domain) binding protein 2 -1.77TOB1 transducer of ERBB2, 1 -1.77UBXN4 UBX domain protein 4 -1.77DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked -1.75FAM29A family with sequence similarity 29, member A -1.75JAK1 Janus kinase 1 (a protein tyrosine kinase) -1.75NFYA nuclear transcription factor Y, alpha -1.75ANP32E acidic (leucine-rich) nuclear phosphoprotein 32 family, member E -1.74EML4 echinoderm microtubule associated protein like 4 -1.74IPO8 importin 8 -1.74MAP3K1 mitogen-activated protein kinase kinase kinase 1 -1.74CDV3 CDV3 homolog (mouse) -1.73DEPDC1 DEP domain containing 1 -1.73PTAR1 protein prenyltransferase alpha subunit repeat containing 1 -1.73RPRD1A regulation of nuclear pre-mRNA domain containing 1A -1.73ZDHHC17 zinc finger, DHHC-type containing 17 -1.73CCDC88A coiled-coil domain containing 88A -1.73ATP13A3 ATPase type 13A3 -1.72CBFB core-binding factor, beta subunit -1.72GABRB3 gamma-aminobutyric acid (GABA) A receptor, beta 3 -1.72MATR3 matrin 3 -1.72PBRM1 polybromo 1 -1.72PIP5K1A phosphatidylinositol-4-phosphate 5-kinase, type I, alpha -1.72PREPL prolyl endopeptidase-like -1.72RC3H2 ring finger and CCCH-type zinc finger domains 2 -1.72RDX radixin -1.72TEX15 testis expressed 15 -1.72CANX calnexin -1.69CCDC88A coiled-coil domain containing 88A -1.69FAF1 Fas (TNFRSF6) associated factor 1 -1.69SCAMP1 secretory carrier membrane protein 1 -1.69APOOL apolipoprotein O-like -1.68ELOVL2 elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3)-like 2 -1.68GDAP1 ganglioside-induced differentiation-associated protein 1 -1.68GLS glutaminase -1.68HECTD1 HECT domain containing 1 -1.68RAB35 RAB35, member RAS oncogene family -1.68DUSP6 dual specificity phosphatase 6 -1.67HIPK1 homeodomain interacting protein kinase 1 -1.67KIF5B kinesin family member 5B -1.67MTMR1 myotubularin related protein 1 -1.67SRPR signal recognition particle receptor (docking protein) -1.67TIA1 TIA1 cytotoxic granule-associated RNA binding protein -1.67TMPO thymopoietin -1.67ZNF146 zinc finger protein 146 -1.67CASC5 cancer susceptibility candidate 5 -1.66CNTN1 contactin 1 -1.66
CRKL v-crk sarcoma virus CT10 oncogene homolog (avian)-like -1.66FNDC3A fibronectin type III domain containing 3A -1.66MAT2A methionine adenosyltransferase II, alpha -1.66UTRN utrophin -1.66CDC2L5 cell division cycle 2-like 5 (cholinesterase-related cell division controller) -1.65CTDSPL2 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small -1.65ETNK1 ethanolamine kinase 1 -1.65HNRNPU heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A -1.65MBTPS2 membrane-bound transcription factor peptidase, site 2 -1.65NARG2 NMDA receptor regulated 2 -1.65RBM14 RNA binding motif protein 14 -1.65TMEM194A transmembrane protein 194A -1.65Sep-11 septin 11 -1.64CACNA2D1 calcium channel, voltage-dependent, alpha 2/delta subunit 1 -1.64G2E3 G2/M-phase specific E3 ubiquitin ligase -1.64NAMPT nicotinamide phosphoribosyltransferase -1.64SNX13 sorting nexin 13 -1.64ADAM10 ADAM metallopeptidase domain 10 -1.6BAG4 BCL2-associated athanogene 4 -1.6HSPH1 heat shock 105kDa/110kDa protein 1 -1.6KCTD20 potassium channel tetramerisation domain containing 20 -1.6MBNL1 muscleblind-like (Drosophila) -1.6RORB RAR-related orphan receptor B -1.6
Upregulated genesSTMN4 stathmin-like 4 2.3CREB5 cAMP responsive element binding protein 5 2.28HINT3 histidine triad nucleotide binding protein 3 1.92JUN jun oncogene 1.87IDH2 isocitrate dehydrogenase 2 (NADP+), mitochondrial 1.83DAAM1 dishevelled associated activator of morphogenesis 1 1.78PSPH phosphoserine phosphatase 1.73AMOTL2 angiomotin like 2 1.72METT10D methyltransferase 10 domain containing 1.67RAP2C RAP2C, member of RAS oncogene family 1.67C4orf30 chromosome 4 open reading frame 30 1.66DUSP10 dual specificity phosphatase 10 1.64
Upregulated genes
Gene symbol Gene nameFold
changeHINT3 histidine triad nucleotide binding protein 3 1.91
SMA4 glucuronidase, beta pseudogene 1.89
SLITRK6 SLIT and NTRK-like family, member 6 1.77
DAAM1 dishevelled associated activator of morphogenesis 1 1.73
JUN jun oncogene 1.68
C21orf45 chromosome 21 open reading frame 45 1.66
PSPH phosphoserine phosphatase 1.65
GRIA2 glutamate receptor, ionotropic, AMPA 2 1.64
C15orf40 chromosome 15 open reading frame 40 1.64
LYZ lysozyme (renal amyloidosis) 1.64
Table 2. Deregulated genes in PrP106–126-treated cells
Table 2
In this paper we have analyzed the monocyte-mediated gene expression profile induced by the amyloid an prion peptides in the human SH-SY5Y neuroblastoma cell line. Results show a relevant coincidence in activating the expression of four genes Hint3, Psph, Daam1 and c-Jun. Furthermore, c-Jun appears to be involved in the cell death mediated by both peptides.
*Research Highlights
Answers to reviewer 2 As requested, we have now included those results not illustrated or mentioned as not shown in the former manuscript. They are shown in the new figures 1 and 5, and a short description has been added into the text and legends. - As indicated in our previous version “the effect was not induced by scrambled
sequences and was not observed with doses of A25-35 or PrP106-126 lower
5M (data not shown)”. These results have been now included in figure 1A, although because their extension in the new figure we have just included the
results corresponding to the higher dose of those compound, that is 5M. Of course, if you consider it is necessary, we could also include the results
obtained with the other doses tested (1 and 2 M), that of course were also unable to reduce cell viability. - In addition, the results obtained in the MTT assay have been included in figure 5. Now it is possible to observe how the reduction of cell viability induced by both fragments is partially reversed in cells transfected with the c-Jun siRNA, thus suggesting a role for Jun in those processes.
*Detailed Response to Reviewers